Ligand Displaces Heat Shock Protein 90 from Overlapping Binding Sites within the Aryl Hydrocarbon Receptor Ligand-binding Domain

Anatoly Soshilov,Michael S Denison

doi:10.1074/jbc.m111.246439

Abstract

Hsp90 (heat shock protein of 90 kDa) is often found associated with functional domains of client proteins, including those for ligand binding, dimerization, DNA binding, and enzymatic activity. Although Hsp90 can maintain the conformation of functionally important domains prior to activation of the client protein, its specific binding site and the mechanism(s) of Hsp90 dissociation during activation are unknown. Here, we have identified and characterized residues involved in Hsp90 binding within the aryl hydrocarbon receptor (AhR) ligand-binding domain and demonstrate that they overlap with those involved in ligand binding. In agreement with this spatial model, ligand binding results in Hsp90 dissociation from the AhR Per-ARNT-Sim B fragment. Interestingly, whereas Hsp90-binding residues within the ligand-binding domain were not involved in Hsp90-dependent AhR protein stability, several of these residues are important for ligand-dependent AhR activation, and their mutation resulted in conversion of two AhR antagonists/partial agonists into full AhR agonists. These studies reveal co-localization of a tentative Hsp90-binding site with that for AhR ligand binding and provide the first molecular mechanism for Hsp90 dissociation in the activation of a client protein.

Highlights

Hsp90 binds to functional domains of protein clients, including the ligand-binding domain of the aryl hydrocarbon receptor (AhR)
Our preliminary results using in vitro synthesized AhR revealed that a single mutation in the AhR Per-ARNT-Sim B (PASB) ligand-binding domain (LBD), K284A, dramatically increased the AhR transformation/DNA binding level (to 148% of that obtained with wtAhR), suggesting that an Hsp90-binding site may be located nearby
The region of the AhR PASB domain encompassing this amino acid is rich in aromatic, basic, and threonine residues (Fig. 1A), a characteristic similar to a region in the Plk1 kinase domain that contains two previously reported natural mutations (P509A and R512W) that eliminate its Hsp90 binding [6]. Utilizing these observations as a guide for site-directed mutagenesis and functional analysis, we attempted to identify and characterize amino acid residues involved in Hsp90 binding contained within the PASB LBD of the AhR

Summary

Background

Hsp binds to functional domains of protein clients, including the ligand-binding domain of the aryl hydrocarbon receptor (AhR). Hsp can maintain the conformation of functionally important domains prior to activation of the client protein, its specific binding site and the mechanism(s) of Hsp dissociation during activation are unknown. Whereas Hsp90-binding residues within the ligand-binding domain were not involved in Hsp90dependent AhR protein stability, several of these residues are important for ligand-dependent AhR activation, and their mutation resulted in conversion of two AhR antagonists/partial agonists into full AhR agonists. These studies reveal co-localization of a tentative Hsp90-binding site with that for AhR ligand binding and provide the first molecular mechanism for Hsp dissociation in the activation of a client protein. During ligand-dependent conversion of the AhR into its high affinity DNA-binding form (referred to as AhR transformation), ligand stimulates AhR nuclear localization through unmasking the nuclear localiza-

The abbreviations used are

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION